RESUMEN
Among the diseases transmitted by vectors, there are those caused by viruses named arboviruses (arthropod-borne viruses). In past years, viruses transmitted by mosquitoes have been of relevance in global health, such as Chikungunya (CHIKV), Dengue (DENV), and Zika (ZIKV), which have Aedes aegypti as a common vector, thus raising the possibility of multi-infection. Previous reports have described the general structure of RNA-dependent RNA polymerases termed right-hand fold, which is conserved in positive single-stranded RNA viruses. Here, we report a comparison between sequences and the computational structure of RNA-dependent RNA polymerases from CHIKV, DENV, and ZIKV and the conserved sites to be considered for the design of an antiviral drug against the three viruses. We show that the sequential identity between consensus sequences from CHIKV and DENV is 8.1% and the similarity is 15.1%; the identity between CHIKV and ZIKV is 9.3%, and the similarity is 16.6%; and the identity between DENV and ZIKV is 68.6%, and the similarity is 79.2%. Nevertheless, the structural alignment shows that the root-mean-square deviation (RMSD) measurement value in general structure comparison between CHIKV RdRp and ZIKV RdRp was 1.248 Å, RMSD between CHIKV RdRp and DENV RdRp was 1.070 Å, and RMSD between ZIKV RdRp and DENV RdRp was 1.106 Å. Despite the low identity and similarity of CHIKV sequence with DENV and ZIKV, we show that A, B, C, and E motifs are structurally well conserved. These structural similarities offer a window into drug design against these arboviruses giving clues about critical target sites.
Asunto(s)
Virus Chikungunya/química , Virus del Dengue/enzimología , ARN Polimerasa Dependiente del ARN/química , Proteínas no Estructurales Virales/química , Virus Zika/enzimología , Secuencias de Aminoácidos , Virus Chikungunya/genética , Virus del Dengue/genética , Humanos , Infecciones por Virus ARN/genética , Infecciones por Virus ARN/terapia , ARN Polimerasa Dependiente del ARN/genética , Homología Estructural de Proteína , Proteínas no Estructurales Virales/genética , Virus Zika/genéticaRESUMEN
Helicobacter pylori (H. pylori) is a pathogen that can remain in the stomach of an infected person for their entire life. As a result, this leads to the development of severe gastric diseases such as gastric cancer. In addition, current therapies have several problems including antibiotics resistance. Therefore, new practical options to eliminate this bacterium, and its induced affections, are required to avoid morbidity and mortality worldwide. One strategy in the search for new drugs is to detect compounds that inhibit a limiting step in a central metabolic pathway of the pathogen of interest. In this work, we tested 55 compounds to gain insights into their possible use as new inhibitory drugs of H. pylori glucose-6-phosphate dehydrogenase (HpG6PD) activity. The compounds YGC-1; MGD-1, MGD-2; TDA-1; and JMM-3 with their respective scaffold 1,3-thiazolidine-2,4-dione; 1H-benzimidazole; 1,3-benzoxazole, morpholine, and biphenylcarbonitrile showed the best inhibitory activity (IC50 = 310, 465, 340, 204 and 304 µM, respectively). We then modeled the HpG6PD protein by homology modeling to conduct an in silico study of the chemical compounds and discovers its possible interactions with the HpG6PD enzyme. We found that compounds can be internalized at the NADP+ catalytic binding site. Hence, they probably exert a competitive inhibitory effect with NADP+ and a non-competitive or uncompetitive effect with G6P, that of the compounds binding far from the enzyme's active site. Based on these findings, the tested compounds inhibiting HpG6PD represent promising novel drug candidates against H. pylori.
Asunto(s)
Simulación por Computador , Inhibidores Enzimáticos/farmacología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Helicobacter pylori/enzimología , Vectores Genéticos/metabolismo , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , Helicobacter pylori/efectos de los fármacos , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Recombinantes/aislamiento & purificación , Homología Estructural de ProteínaRESUMEN
Giardia lamblia, due to the habitat in which it develops, requires a continuous supply of intermediate compounds that allow it to survive in the host. The pentose phosphate pathway (PPP) provides essential molecules such as NADPH and ribulose-5-phosphate during the oxidative phase of the pathway. One of the key enzymes during this stage is 6-phosphogluconate dehydrogenase (6 PGDH) for generating NADPH. Given the relevance of the enzyme, in the present work, the 6pgdh gene from G. lamblia was amplified and cloned to produce the recombinant protein (Gl-6 PGDH) and characterize it functionally and structurally after the purification of Gl-6 PGDH by affinity chromatography. The results of the characterization showed that the protein has a molecular mass of 54 kDa, with an optimal pH of 7.0 and a temperature of 36-42 °C. The kinetic parameters of Gl-6 PGDH were Km = 49.2 and 139.9 µM (for NADP+ and 6-PG, respectively), Vmax =26.27 µmol*min-1*mg-1, and Kcat = 24.0 s-1. Finally, computational modeling studies were performed to obtain a structural visualization of the Gl-6 PGDH protein. The generation of the model and the characterization assays will allow us to expand our knowledge for future studies of the function of the protein in the metabolism of the parasite.
Asunto(s)
Giardia lamblia/enzimología , Gluconatos/química , NADP/química , Fosfogluconato Deshidrogenasa/química , Proteínas Protozoarias/química , Ribulosafosfatos/química , Secuencias de Aminoácidos , Sitios de Unión , Clonación Molecular/métodos , Expresión Génica , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/enzimología , Giardia lamblia/genética , Gluconatos/metabolismo , Humanos , Cinética , Modelos Moleculares , NADP/metabolismo , Vía de Pentosa Fosfato/genética , Fosfogluconato Deshidrogenasa/genética , Fosfogluconato Deshidrogenasa/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulosafosfatos/metabolismo , Homología Estructural de Proteína , Especificidad por Sustrato , TermodinámicaRESUMEN
Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.
Asunto(s)
Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Mutantes/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Benzotiazoles/química , Benzotiazoles/metabolismo , Sitios de Unión , AMP Cíclico/química , AMP Cíclico/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Ligandos , Dominios Proteicos , Homología Estructural de Proteína , Termodinámica , Triazoles/química , Triazoles/metabolismoRESUMEN
Several antidepressants inhibit nicotinic acetylcholine receptors (nAChRs) in a non-competitive and voltage-dependent fashion. Here, we asked whether antidepressants with a different structure and pharmacological profile modulate the rat α7 nAChR through a similar mechanism by interacting within the ion-channel. We applied electrophysiological (recording of the ion current elicited by choline, ICh, which activates α7 nAChRs from rat CA1 hippocampal interneurons) and in silico approaches (homology modeling of the rat α7 nAChR, molecular docking, molecular dynamics simulations, and binding free energy calculations). The antidepressants inhibited ICh with the order: norfluoxetine ~ mirtazapine ~ imipramine < bupropion ~ fluoxetine ~ venlafaxine ~ escitalopram. The constructed homology model of the rat α7 nAChR resulted in the extracellular vestibule and the channel pore is highly negatively charged, which facilitates the permeation of cations and the entrance of the protonated form of antidepressants. Molecular docking and molecular dynamics simulations were carried out within the ion-channel of the α7 nAChR, revealing that the antidepressants adopt poses along the receptor channel, with slightly different binding-free energy values. Furthermore, the inhibition of ICh and free energy values for each antidepressant-receptor complex were highly correlated. Thus, the α7 nAChR is negatively modulated by a variety of antidepressants interacting in the ion-channel.
Asunto(s)
Antidepresivos/química , Antidepresivos/farmacología , Canales Iónicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Antidepresivos/clasificación , Colina/farmacología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Ratas , Homología Estructural de Proteína , Relación Estructura-Actividad , TermodinámicaRESUMEN
Lectins are ubiquitous proteins involved in the immune defenses of different organisms and mainly responsible for non-self-recognition and agglutination reactions. This work describes molecular and biological characterization of a rhamnose-binding lectin (RBL) from Rhodnius prolixus, which possesses a 21 amino acid signal peptide and a mature protein of 34.6 kDa. The in-silico analysis of the primary and secondary structures of RpLec revealed a lectin domain fully conserved among previous insects studied. The three-dimensional homology model of RpLec was similar to other RBL-lectins. Docking predictions with the monosaccharides showed rhamnose and galactose-binding sites comparable to Latrophilin-1 and N-Acetylgalactosamine-binding in a different site. The effects of RpLec gene silencing on levels of infecting Trypanosoma cruzi Dm 28c and intestinal bacterial populations in the R. prolixus midgut were studied by injecting RpLec dsRNA into the R. prolixus hemocoel. Whereas T. cruzi numbers remained unchanged compared with the controls, numbers of bacteria increased significantly. The silencing also induced the up regulation of the R. prolixus defC (defensin) expression gene. These results with RpLec reveal the potential importance of this little studied molecule in the insect vector immune response and homeostasis of the gut bacterial microbiota.
Asunto(s)
Enfermedad de Chagas/inmunología , Defensinas/administración & dosificación , Microbioma Gastrointestinal/genética , Proteínas de Insectos/genética , Lectinas/metabolismo , Rhodnius/fisiología , Trypanosoma cruzi/fisiología , Animales , Defensinas/metabolismo , Vectores de Enfermedades , Proteínas de Peces/genética , Silenciador del Gen , Inmunidad Innata , Proteínas de Insectos/metabolismo , Lectinas/genética , Simulación del Acoplamiento Molecular , ARN Ribosómico 16S/genética , Homología Estructural de ProteínaRESUMEN
Internal structure similarity in proteins can be observed at the domain and subdomain levels. From an evolutionary perspective, structurally similar elements may arise divergently by gene duplication and fusion events but may also be the product of convergent evolution under physicochemical constraints. The characterization of proteins that contain repeated structural elements has implications for many fields of protein science including protein domain evolution, structure classification, structure prediction, and protein engineering. FiRES (Find Repeated Elements in Structure) is an algorithm that relies on a topology-independent structure alignment method to identify repeating elements in protein structure. FiRES was tested against two hand curated databases of protein repeats: MALIDUP, for very divergent duplicated domains; and RepeatsDB for short tandem repeats. The performance of FiRES was compared to that of lalign, RADAR, HHrepID, CE-symm, ReUPred, and Swelfe. FiRES was the method that most accurately detected proteins either with duplicated domains (accuracy = 0.86) or with multiple repeated units (accuracy = 0.92). FiRES is a new methodology for the discovery of proteins containing structurally similar elements. The FiRES web server is publicly available at http://fires.ifc.unam.mx. The scripts, results, and benchmarks from this study can be downloaded from https://github.com/Claualvarez/fires.
Asunto(s)
Algoritmos , Proteínas/química , Programas Informáticos , Homología Estructural de Proteína , Secuencia de Aminoácidos , Benchmarking , Bases de Datos de Proteínas , Evolución Molecular , Duplicación de Gen , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. METHODS: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. FINDINGS: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. INTERPRETATION: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms. FUNDING: This work was supported by grants from CNPq and FAPERJ-Brazil.
Asunto(s)
Neoplasias Esofágicas/enzimología , Neoplasias Esofágicas/genética , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Anciano , Secuencia de Aminoácidos , Biomarcadores de Tumor/metabolismo , Neoplasias Esofágicas/diagnóstico , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Persona de Mediana Edad , Modelos Moleculares , Subunidades de Proteína/química , Subunidades de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Curva ROC , Homología Estructural de Proteína , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Bacterial chemoreceptors control the activity of the associated CheA kinase in response to chemical gradients and, consequently, regulate the swimming behavior of the cell. However, such control is not direct but requires the participation of the essential coupling protein CheW, which is structurally homologous to the carboxy-terminal domain of the kinase. The actual role of this small coupling protein is somehow intriguing. It has been demonstrated that it is absolutely essential for chemoreceptor control of the kinase, in spite of the occurrence of direct contacts between chemoreceptors and CheA. In addition, CheW plays an essential role in the assembly of the large macromolecular arrays that combine chemoreceptors of different specificities, and it is therefore responsible for molecular interactions that provide such arrays with remarkable signaling properties. In this work, we analyze truncated CheW derivatives that are still able to control the kinase but have lost the ability to connect signaling units. We demonstrate that these two activities can work separately and speculate about the significance of the roles of these two different activities in the context of the chemoreceptor cluster.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Quimiotaxis , Proteínas de Escherichia coli/ultraestructura , Escherichia coli/ultraestructura , Histidina Quinasa/ultraestructura , Proteínas Quimiotácticas Aceptoras de Metilo/ultraestructura , Proteínas Bacterianas/genética , Sitios de Unión , Quimiotaxis/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Histidina Quinasa/genética , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Transducción de Señal/genética , Homología Estructural de ProteínaRESUMEN
Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.
Asunto(s)
Proteínas Bacterianas/química , Brucella/química , ADN/química , Factores de Virulencia/química , Secuencias de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Brucella/patogenicidad , ADN/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Motivos de Nucleótidos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Homología Estructural de Proteína , Termodinámica , Factores de Virulencia/metabolismoRESUMEN
Homology modeling is a computational approach to generate three-dimensional structures of protein targets when experimental data about similar proteins are available. Although experimental methods such as X-ray crystallography and nuclear magnetic resonance spectroscopy successfully solved the structures of nearly 150,000 macromolecules, there is still a gap in our structural knowledge. We can fulfill this gap with computational methodologies. Our goal in this chapter is to explain how to perform homology modeling of protein targets for drug development. We choose as a homology modeling tool the program MODELLER. To illustrate its use, we describe how to model the structure of human cyclin-dependent kinase 3 using MODELLER. We explain the modeling procedure of CDK3 apoenzyme and the structure of this enzyme in complex with roscovitine.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas/química , Programas Informáticos , Secuencia de Aminoácidos , Diseño de Fármacos , Humanos , Conformación Proteica , Homología Estructural de Proteína , Interfaz Usuario-Computador , Navegador WebRESUMEN
HLA epitope analysis emerged as a strategy to determine alloimmune risk in solid organ transplantation. However, it requires not only knowledge on HLA amino acids sequences, but also on HLA three-dimensional structures. Unfortunately, the number of structures available is still unsatisfactory. This work reports the modelling of 106 heterotrimeric (alpha chainâ¯+â¯ß2Mâ¯+â¯peptide) HLA class I molecules. The models were generated by homology modelling using Modeller, refined using GalaxyRefine server, heterodimerized with Swiss-PDB Viewer and, finally, assessed as to their structural quality through Dali server. The final structures were made available through a free online database, pHLA3D (www.phla3d.com.br), developed in Ruby language using the Ruby on Rails web framework. Structural parameters were similar between refined molecules and their templates. The new database may improve HLA epitope analysis and better guide risk assessment in solid organ transplantation setting.
Asunto(s)
Sistemas de Administración de Bases de Datos , Bases de Datos de Proteínas , Antígenos de Histocompatibilidad Clase I/química , Estructura Secundaria de Proteína , Homología Estructural de Proteína , Alelos , Secuencia de Aminoácidos , Epítopos/inmunología , Histocompatibilidad , Humanos , Modelos Moleculares , Navegador WebRESUMEN
An in silico analysis of the interaction between the complex-ligands of nine acetylcholinesterase (AChE) structures of Lepidopteran organisms and 43 organophosphorus (OPs) pesticides with previous resistance reports was carried out. To predict the potential resistance by structural modifications in Lepidoptera insects, due to proposed point mutations in AChE, a broad analysis was performed using computational tools, such as homology modeling and molecular docking. Two relevant findings were revealed: (1) Docking results give a configuration of the most probable spatial orientation of two interacting molecules (AChE enzyme and OP pesticide) and (2) a predicted ΔGb. The mutations evaluated in the form 1 acetylcholinesterase (AChE-1) and form 2 acetylcholinesterase (AChE-2) structures of enzymes do not affect in any way (there is no regularity of change or significant deviations) the values of the binding energy (ΔGb) recorded in the AChE-OPs complexes. However, the mutations analyzed in AChE are associated with a structural modification that causes an inadequate interaction to complete the phosphorylation of the enzyme.
Asunto(s)
Acetilcolinesterasa/química , Acetilcolinesterasa/genética , Resistencia a los Insecticidas/efectos de los fármacos , Resistencia a los Insecticidas/genética , Lepidópteros/genética , Compuestos Organofosforados/farmacología , Plaguicidas/farmacología , Mutación Puntual/efectos de los fármacos , Animales , Biología Computacional/métodos , Simulación por Computador , Lepidópteros/efectos de los fármacos , Lepidópteros/enzimología , Simulación del Acoplamiento Molecular , Compuestos Organotiofosforados/química , Fragmentos de Péptidos , Fosforamidas/química , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
The Golgi complex is part of the endomembrane system and is responsible for receiving transport cargos from the endoplasmic reticulum and for sorting and targeting them to their final destination. To perform its function in higher eukaryotic cells, the Golgi needs to be correctly assembled as a flattened membrane sandwich kept together by a protein matrix. The precise mechanism controlling the Golgi cisternae assembly is not yet known, but it is widely accepted that the Golgi Reassembly and Stacking Protein (GRASP) is a main component of the Golgi protein matrix. Unlike mammalian cells, which have two GRASP genes, lower eukaryotes present only one gene and distinct Golgi cisternae assembly. In this study, we performed a set of biophysical studies to get insights on the structural properties of the GRASP domains (DGRASPs) from both human GRASP55 and GRASP65 and compare them with GRASP domains from lower eukaryotes (Saccharomyces cerevisiae and Cryptococcus neoformans). Our data suggest that both human DGRASPs are essentially different from each other and that DGRASP65 is more similar to the subgroup of DGRASPs from lower eukaryotes in terms of its biophysical properties. GRASP55 is present mainly in the Golgi medial and trans faces, which are absent in both fungi, while GRASP65 is located in the cis-Golgi. We suggest that the GRASP65 gene is more ancient and that its paralogue GRASP55 might have appeared later in evolution, together with the medial and trans Golgi faces in mammalians.
Asunto(s)
Proteínas Fúngicas/química , Proteínas de la Matriz de Golgi/química , Homología Estructural de Proteína , Cryptococcus neoformans , Evolución Molecular , Proteínas Fúngicas/genética , Proteínas de la Matriz de Golgi/genética , Proteínas de la Matriz de Golgi/metabolismo , Saccharomyces cerevisiaeRESUMEN
Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.
Asunto(s)
Corynebacterium pseudotuberculosis/metabolismo , Vitamina B 12/metabolismo , Adenina/química , Adenina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cinética , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Estructura Secundaria de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Espectrometría de Fluorescencia , Homología Estructural de Proteína , Suramina/química , Suramina/metabolismo , Vitamina B 12/biosíntesis , Vitamina B 12/químicaRESUMEN
The present study gives an overview of the binding energetics of the homologous heterodimers of cruzipain-chagasin based on the binding energy (ΔGb) prediction obtained with FoldX. This analysis involves a total of 70 homologous models of the cruzipain-chagasin complex which were constructed by homology from the combinatory variation of nine papain-like cysteine peptidase structures and seven cysteine protease inhibitor structures (as chagasin-like and cystatin-like inhibitors). Only 32 systems have been evaluated experimentally, ΔGbexperimental values previously reported. Therefore, the result of the multiple analysis in terms of the thermodynamic parameters, are shown as relative energy |ΔΔG| = |ΔGbfrom FoldX - ΔGbexperimental|. Nine models were identified that recorded |ΔΔG| < 1.3, five models to 2.8 > |ΔΔG| > 1.3 and the other 18 models, values of |ΔΔG| > 2.8. The energetic analysis of the contribution of ΔH and ΔS to ΔGb to the 14-molecular model presents a ΔGb mostly ΔH-driven at neutral pH and at an ionic strength (I) of 0.15 M. The dependence of ΔGb(I,pH) at 298 K to the cruzipain-chagasin complex predicts a linear dependence of ΔGb(I). The computational protocol allowed the identification and prediction of thermodynamics binding energy parameters for cruzipain-chagasin-like heterodimers.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multiproteicos/química , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Cisteína Endopeptidasas/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Protozoarias/química , Homología Estructural de ProteínaRESUMEN
One of the most widespread pathogens worldwide is methicillin-resistant Staphylococcus aureus, a bacterium that provokes severe life-threatening illnesses both in hospitals and in the community. The principal challenge lies in the resistance of MRSA to current treatments, which encourages the study of different molecular targets that could be used to develop new drugs against this infectious agent. With this goal, a detailed characterization of shikimate kinase from this microorganism (SaSK) is described. The results showed that SaSK has a Km of 0.153 and 224 µM for shikimate and ATP, respectively, and a global reaction rate of 13.4 µmol/min/mg; it is suggested that SaSK utilizes the Bi-Bi Ping Pong reaction mechanism. Furthermore, the physicochemical data indicated that SaSK is an unstable, hydrophilic, and acidic protein. Finally, structural information showed that SaSK presented folding that is typical of its homologous counterparts and contains the typical domains of this family of proteins. Amino acids that have been shown to be important for SaSK protein function are conserved. Therefore, this study provides fundamental information that may aid in the design of inhibitors that could be used to develop new antibacterial agents.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenosina Trifosfato/metabolismo , Diseño de Fármacos , Estabilidad de Enzimas , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Ácido Shikímico/metabolismo , Homología Estructural de ProteínaRESUMEN
BACKGROUND: The polyamine oxidases (PAOs) catabolize the oxidative deamination of the polyamines (PAs) spermine (Spm) and spermidine (Spd). Most of the phylogenetic studies performed to analyze the plant PAO family took into account only a limited number and/or taxonomic representation of plant PAOs sequences. RESULTS: Here, we constructed a plant PAO protein sequence database and identified four subfamilies. Subfamily PAO back conversion 1 (PAObc1) was present on every lineage included in these analyses, suggesting that BC-type PAOs might play an important role in plants, despite its precise function is unknown. Subfamily PAObc2 was exclusively present in vascular plants, suggesting that t-Spm oxidase activity might play an important role in the development of the vascular system. The only terminal catabolism (TC) PAO subfamily (subfamily PAOtc) was lost in Superasterids but it was present in all other land plants. This indicated that the TC-type reactions are fundamental for land plants and that their function could being taken over by other enzymes in Superasterids. Subfamily PAObc3 was the result of a gene duplication event preceding Angiosperm diversification, followed by a gene extinction in Monocots. Differential conserved protein motifs were found for each subfamily of plant PAOs. The automatic assignment using these motifs was found to be comparable to the assignment by rough clustering performed on this work. CONCLUSIONS: The results presented in this work revealed that plant PAO family is bigger than previously conceived. Also, they delineate important background information for future specific structure-function and evolutionary investigations and lay a foundation for the deeper characterization of each plant PAO subfamily.
Asunto(s)
Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Plantas/enzimología , Análisis de Secuencia de Proteína , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Análisis por Conglomerados , Bases de Datos de Proteínas , Filogenia , Dominios Proteicos , Homología Estructural de Proteína , Poliamino OxidasaRESUMEN
The WFDC1 gene is frequently down-regulated or lost in prostate cancer, and the encoded protein, ps20, has been implicated in epithelial cell behaviour and angiogenesis. However, ps20 remains largely uncharacterised with respect to its structure and interacting partners. This study characterised the evolution, functionality and structural characteristics of WFDC1/ps20 using phylogenetic reconstruction and other computational approaches. Bayesian phylogenetic analyses suggested that ps20 appeared in a common ancestor of deuterostomes-protostomes. The rate of evolutionary change within the coding regions of vertebrate WFDC1 genes and the synteny conservation in mammals differed from that of other vertebrate clades, indicating a possible functional diversity of ps20 homologues. A gene set enrichment analysis of the genes around WFDC1 (conserved synteny) showed functional relationships between the WFDC1, CDH13, CRISPLD2, IRF8 and TFPI2 genes. The molecular evolution of ps20 has been driven by purifying selection, particularly in the segments corresponding to exons 3 and 4, which encode the most conserved regions of the protein. A co-evolution analysis showed that residues within these regions co-vary with each other during the evolution of ps20. These results show that the regions corresponding to exons 3 and 4 are ps20-specific structure-function modules. Homology modelling of the exon 2-encoded polypeptide and subsequent dynamics calculus using a Gaussian network model showed that residues with high conformational flexibility are part of a loop region involved in protein-protein recognition, given the similarity with other serine protease inhibitors. Residues C96, R94, L105, and C66 are critical for the integrity and functionality of this ps20 region.
Asunto(s)
Evolución Molecular , Modelos Moleculares , Filogenia , Proteínas , Humanos , Dominios Proteicos , Proteínas/química , Proteínas/genética , Homología Estructural de ProteínaRESUMEN
l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 µM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.